Role of hyperglycemia in isogeneic islet transplantation: an experimental animal study.

OBJECTIVE: Study the role of hyperglycemia-induced beta cell loss on grafted islet destruction. DESIGN: Male inbred rats were made diabetic by streptozotocin administration and used as islet donors and/or isograft recipients to probe directly the role of hyperglycemia as an important determinant of transplanted islet fate, following exclusion of immune-related causes of islet graft destruction like allograft immunity and disease recurrence. RESULTS: Our studies showed that: a) Hyperglycemia destroyed islet but not pituitary isografts and b) Tight control of normoglycemia by sufficient islet mass engraftment prevented graft damage. CONCLUSION: While sustained hyperglycemia caused destruction of transplanted islet isografts, induction of normoglycemia by transplantation of sufficient islet mass to diabetic recipients had a beneficial long term effect on their functional engraftment.

Association of an oversized adrenal cortical adenoma with expression of pheochromocytoma-like neurosecretory features.

Abnormal stimulation of adrenal function may be either direct, affecting similarly cortical and medullary secretion, or indirect affecting primarily the medulla. Indirect activation of clinically detectable adrenomedullary function may develop as a physical consequence of a non-functional adrenal tumor exerting pressure on the medulla by its size, location and direction of growth. Our case of an oversized and overweight adrenal tumor associated with expression of late-onset pheochromocytoma-like clinical symptoms may be explained by the physical indirect rather than the biological direct activation of adrenomedullary function like hyperplasia or cancer.

Evidence of direct estrogenic regulation of human corticotropin-releasing hormone gene expression. Potential implications for the sexual dimophism of the stress response and immune/inflammatory reaction.

Corticotropin-releasing hormone (CRH) plays major roles in coordination of the stress response and regulation of the immune/inflammatory reaction, two important functions associated with sexual dimorphism. Two overlapping segments of the 5' flanking region of the human (h) CRH gene, the proximal 0.9 kb (containing two perfect half-palindromic estrogen-responsive elements [EREs]) and the 2.4 kb (including the former and containing two additional perfect half-palindromic EREs), were examined for their ability to confer estrogen-mediated transcriptional enhancement to a homologous or heterologous promoter. The level of estrogen-induced transactivation by the 0.9- and 2.4-kb segments was determined by chloramphenicol acetyltransferase analysis in CV-1 cells cotransfected with estrogen receptor (ER) cDNA expression plasmids, and found to be respectively approximately 10% and 20% of that of the strongly estrogen-responsive Xenopus vitellogenin A2 enhancer. Gel retardation and immunoprecipitation demonstrated specific association between the perfect half-palindromic EREs of hCRH gene and the DNA binding domain of hER in vitro. These findings may constitute the basis of sexual dimorphism in the expression of the CRH gene in the central nervous system and periphery, and might shed light in existing gender differences in stress response and immune regulation.

Reevaluating pancreatic duct ligation in Whipple procedure.

In an effort to avoid the morbidity and mortality related to pancreaticojejunal anastomosis after pancreaticoduodenectomy (PPD), we report on the treatment of the pancreatic stump by pancreatic duct ligation (PDL) following Whipple procedure. We studied a series of 9 consecutive unselected patients (8 with pancreatic cancer and 1 with chronic pancreatitis). Of those, pancreatic fistula occurred in 4 patients and persisted for 14 to 58 days (mean 35.4 days). Two patients died within 30 days after surgery from causes not related to PDL. None of our patients developed diabetes mellitus following PDL surgery, nor any of the other frequently mentioned postoperative complications such as acute pancreatitis or pancreatic insufficiency. In conclusion, PDL may occasionally lead to a controlled pancreaticocutaneous fistula with fading inactive contents over time not causing further metabolic complications but is a safe, simple and fast alternative to pancreaticojejunostomy.

Structural analysis of the regulatory region of the human corticotropin releasing hormone gene.

A DNA fragment containing the human corticotropin releasing hormone (CRH) gene, along with 9 kb of upstream and 4 kb of downstream sequences, was isolated from a human genomic DNA library. Nucleotide sequence analysis of the proximal 918 nucleotides 5' flanking the putative major mRNA start site of the human gene and comparison to the 866 nucleotide long homologous ovine sequence, revealed that this region of the CRH gene consists of two distinct areas with different degrees of homology, varying from 72% to 94%. The putative functional features of the human sequence were identified. Many, but not all, features were conserved in the ovine sequence. The highly conserved nature of the regulatory region of this gene makes it a good candidate for tracing possible related genetic defects of the hypothalamic-pituitary-adrenal (HPA) axis.

Mapping interleukin enhancer binding factor 2 gene (ILF2) to human chromosome 1 (1q11-qter and 1p11-p12) by polymerase chain reaction amplification of human-rodent somatic cell hybrid DNA templates.

Interleukin-2 (IL-2) is the first lymphokine secreted following T cell activation. Several transcription factors regulate IL-2 gene expression, including the nuclear factor of activated T cells (NFAT). NFAT acts at the antigen receptor response element-2 (ARRE-2) sequence in the IL-2 enhancer and is the nuclear target of T cell stimulation signals and the immunosuppressant drugs cyclosporine and FK506, which are potent inhibitors of IL-2 gene transcription. NFAT has been cloned and found to consist of two subunits, NF45 (ILF2) and NF90 (ILF3). This communication reports the assignment of NF45, interleukin enhancer binding factor 2 gene (ILF2), to human chromosome 1 (1q11-qter and 1p11-p12) by polymerase chain reaction (PCR) amplification of ILF2-specific DNA sequences from well-characterized human-rodent somatic cell hybrid DNA.

Mapping interleukin enhancer binding factor 3 gene (ILF3) to human chromosome 19 (19q11-qter and 19p11-p13.1) by polymerase chain reaction amplification of human-rodent somatic cell hybrid DNA templates.

Interleukin-2 (IL-2) is the first lymphokine secreted following T cell activation. Several transcription factors regulate IL-2 gene expression, including the nuclear factor of activated T cells (NFAT). NFAT acts at the antigen receptor response element-2 (ARRE-2) sequence in the IL-2 enhancer and is the nuclear target of T cell stimulation signals and the immunosuppressant drugs cyclosporine (Sandimmune) and FK-506 (tacrolimus), which are potent inhibitors of IL-2 gene transcription. NFAT has been cloned and found to consist of two subunits, NF45 (ILF2) and NF90 (ILF3). We have recently assigned the gene encoding the small NFAT subunit, NF45 (ILF3) to human chromosome 1 (1q11-qter and 1p11-p12). This communication reports the assignment of the gene encoding the large NFAT subunit, NF90 or interleukin enhancer binding factor 3 gene (ILF3), to human chromosome 19 (19q11-qter and 19p11-p13.1) by polymerase chain reaction (PCR) amplification of ILF3-specific DNA sequences from well-characterized human-rodent somatic cell hybrid DNAs.

Regulated activity of the distal promoter-like element of the human corticotropin-releasing hormone gene and secondary structural features of its corresponding transcripts.

Corticotropin-releasing hormone (CRH) plays a major role in the coordination of the stress response. Its gene is expressed in multiple brain regions, the peripheral sympathetic system and the placenta, as well as in peripheral inflammatory sites where CRH acts as a pro-inflammatory cytokine. The human (h) CRH gene, in addition to its primary promoter (TATA box I), has a second distal promoter-like structure (TATA box II) and a functional cyclic adenosine monophosphate-responsive element, all of which are preserved in the rat and ovine genes. To examine the functionality of TATA II, we positioned a 881-bp-long segment of the 5' flanking region of the hCRH gene containing TATA II, but lacking TATA I, upstream from a chloramphenicol acetyltransferase (CAT) reporter gene cloned in a pUC vector. We transfected COS-7 cells with this construct and examined responsiveness of CAT activity to potential stimulants and inhibitors. Phorbol ester (TPA) and forskolin had mild but clear stimulatory effects on CAT expression (approximately 1.5- and approximately 1.3-fold, respectively), with a combined effect of approximately 1.9-fold. Dexamethasone (DEX) inhibited TPA-stimulated CAT activity by approximately 2.6-fold. In contrast, in the presence of a co-transfected glucocorticoid receptor cDNA expression plasmid, DEX augmented TPA-stimulated CAT expression by approximately 3.1-fold. The predicted secondary structures of the primary transcripts employing the distal and proximal promoters had significant differences, which could affect their stability and translatability.2

Heroin-induced changes of catecholamine-containing particles in male rat cerebellar cortex.

The content and distribution of catecholamine-containing formations in the cerebellum of untreated and heroin-treated male rats, was visualized by glyoxylic acid-induced histofluorescence, in an attempt to define the adaptive mechanisms leading to heroin dependent tolerance as well as identify a biological role for these formations. Repeated heroin administration increased the number of specifically organized intracellular catecholamine containing particles, including grain (diameter less than 0.8 microm) and aggregate (diameter greater than 1 microm) forms, in all cerebellar cortical layers examined one hour after the last injection of the drug, relative to controls. The number of grains in all cerebellar cortical layers examined and aggregates in the granular layer, returned to normal or near normal baseline levels within twenty four hours after the last injection of the drug. The analogous baseline of the aggregates in the Purkinje cell layer primarily and the Molecular layer secondarily remained significantly elevated by 86% and 50% respectively, relative to controls. Catecholamine-heroin interactions most likely mediated this elevation that was related directly to the heroin-dependent state of tolerance. These findings indicate that heroin administration to heroin-tolerant rats leads to the formation of unusually large intracellular aggregates with catecholamines in the Purkinje cells of the cerebellum primarily and support a direct role for these formations in the modulation of biogenic amine bioavailability. We conclude that adaptation to drug exposure involves multiple homeostatic interactions, with sympathetic activation at the level of catecholamine reorganization and redistribution playing a major role in rat cerebellar cortex.

Lack of dexamethasone modulation of mRNAs involved in the glucocorticoid signal transduction pathway in two cell systems.

Glucocorticoids are final effectors of the stress response. These hormones exert negative feedback action at multiple levels of the hypothalamic-pituitary-adrenal axis and regulate a large number of central nervous system and peripheral target genes. The inactive form of the glucocorticoid receptor in the cytoplasm appears to be bound to heat shock proteins of the 90K family (hsp90 alpha and hsp90 beta). This interaction facilitates binding of glucocorticoid to its receptor and, therefore, its activation. The chicken ovalbumin upstream promoter transcription factor (COUP-TF) binds on a negative glucocorticoid response element in the 5' regulatory region of the proopiomelanocortin gene and prevents the repressive effect of glucocorticoids on this gene. The aim of this study was to examine the effect of glucocorticoids on the steady-state mRNAs of their own receptor, the two hsp90s, and COUP-TF. Quantitative Northern blot analysis in primary leukocytes and Epstein-Barr virus-transformed human lymphocytes (EBV-THL) basally and after a 24-hour exposure to 50 nM dexamethasone was performed. Treatment of primary leukocytes or normally growing EBV-THL with dexamethasone had no effect on the mRNA level of glucocorticoid receptor, hsp90 alpha, hsp90 beta, or COUP-TF. Similar treatment of EBV-THL grown in charcoal-stripped media, resulted in minimal changes in the mRNAs of these factors. Our findings suggest that glucocorticoids do not regulate the steady-state mRNA levels of these core components of the mammalian stress response in human primary and Epstein-Barr virus-transformed lymphocytes.

Correlating functional staging to effective treatment of acute surgical illness.

BACKGROUND: Proinflammatory and anti-inflammatory events may eventually trigger host response, which acting via a broad spectrum of complex biological processes and molecular interactions may either enhance or resolve the symptoms of acute surgical illness (ASI). Staging the sequence of biological events that take place at the cellular level during the development of ASI may provide leads to effective stage-specific treatments. In line with the hypothesis that proper timing of therapeutic intervention may be crucial to the management of the disease, we have attempted in this review to correlate functional staging to effective treatment of ASI. DATA SOURCE: The present report proposes a conceptual synthesis on the biogenesis and treatment of ASI that is based on known molecular and cellular aspects of human inflammatory sequence and patient data from clinical trials. It also introduces proper timing of therapeutic intervention as a potentially important determinant for the successful outcome of the disease process. CONCLUSIONS: Progress in understanding the biogenesis of ASI did not result in successful therapeutic developments as yet. The challenge ahead should be a better understanding of the dynamics of the various processes and regulators in appropriate animal and clinical models of ASI, in order to properly intervene and direct effective therapies for the benefit of critically ill patients.

Improved detection of Dirofilaria repens DNA by direct polymerase chain reaction.

Diagnosis of human infection by Dirofilaria repens, depends mainly on microscopic evaluation of tissue cross-sections and the macroscopic characteristics of the worm. Tissue degeneration and/or poor specimen preparation practices however, often render many cases of subcutaneous dirofilariasis elusive to such morphological diagnostic approaches. The early PCR protocols, developed to satisfy these complex diagnostic needs, failed to amplify dirofilariae DNA from formalin preserved material. To overcome these difficulties, we developed an improved PCR protocol using a set of primers designed to amplify a rather stable, highly repetitive D. repens-specific genomic DNA target. We report the performance of this protocol with a large variety of dirofilariae infected DNA specimens, including those extracted from formalin preserved biological material for up to 20 days. Our findings support its potential application to routine clinical diagnosis.

Structural organization of the 5' flanking region of the human corticotropin releasing hormone gene.

We have determined the nucleotide sequence of the proximal 3625 nucleotides 5' flanking the major mRNA start site of the human corticotropin releasing hormone gene (hCRH) and identified several putative regulatory elements. Interestingly, we did not detect any glucocorticoid responsive elements; we did however find five interspersed perfect half palindromic estrogen responsive elements, which might confer estrogen regulatability to the hCRH gene. We have identified a segment spanning from -2835 to -2972, which has about 72% homology to the 3' terminal half of the human Alu I family of highly repetitive elements, and another one, which spans from -2213 to -2580 and has greater than 80% homology to members of human type O family of repetitive elements. These elements may confer DNA fragility, since the loci for hCRH and the human fragile site FRA8F colocalize in human chromosome 8. The structural information reported represents a first step in the study of regulation of the hCRH gene at the molecular level.

Surgical complications of hyperglycaemia.

We review the mechanisms leading to hyperglycaemic damage and draw functional extrapolations aiming to an improved management of surgical complications, which are common among diabetic patients.

Comparing normal primary endocervical adenoepithelial cells to uninfected and influenza B virus infected human cervical adenocarcinoma HeLa cells.

Human adenocarcinoma HeLa cells surviving infection with low (10(-9) units), medium (10(-6) units), and high (10(-2) units) influenza B titers were compared to their uninfected precursors and to normal endocervical adenoepithelial and metaplastic cells using Papanikolaou-staining method and immunocytochemistry. Normal primary endocervical and infected HeLa cells surviving infection shared similar morphologic, phenotypic, and divisional patterns that differed drastically from those of uninfected HeLa cells. The number of infected hosts surviving 6-7 days of viral exposure did not change during 3-week follow-up period, and their cyclin E levels suggested that they had been arrested to the G1 phase of the cell cycle by viral stress. Our findings suggest that in addition to apoptosis, nononcogenic viral stress activated the expression of endocervical metaplastic-like motifs in surviving hosts. A mechanism of cell response to nononcogenic viral stress was proposed to explain these findings. We conclude that nononcogenic respiratory viruses specifically target and eliminate abnormal cells ectopically overexpressing appropriate receptors and may complement current treatments of cervical cancer.

Human corticotropin-releasing hormone receptor gene (CRHR) is located on the long arm of chromosome 17 (17q12-qter).

Using polymerase chain reaction amplification of commercially available DNA templates, we have mapped the human corticotropin-c releasing hormone receptor gene (CRHR) to the long arm of chromosome 17 (17q12-qter).

Identification of separate mRNAs coding for the alpha and beta subunits of thyrotropin.

A 9S mRNA, purified from mouse thyrotropic pituitary tumors by sucrose density gradient centrifugation of poly(A)-enriched mRNA, directed the synthesis of only alpha and beta subunits of thyrotropin in the reticulocyte lysate translation system. Analysis of radioiodinated 9S mRNA, repurified by oligo(dT)-cellulose chromatography and sucrose gradient centrifugation, yielded two species of RNA on urea/polyacrylamide gel electrophoresis. The major RNA species contained 620 nucleotides, and the minor RNA species contained 560 nucleotides. Unlabeled 9S mRNA was further purified by urea/polyacrylamide gel electrophoresis; the mRNAs were separately eluted from slices of the gel containing material migrating with an apparent length of 620 and 560 nucleotides. Translation of these mRNAs in the reticulocyte lysate showed that the longer mRNA coded for the alpha subunit and the shorter mRNA coded for the beta subunit of mouse thyrotropin. Because more alpha than beta subunit of thyrotropin was consistently synthesized, unbalanced amounts of thyrotropin subunits appear to be synthesized by translation of unbalanced amounts of individual mRNAs. We have demonstrated that the synthesis of thyrotropin is directed by two separate mRNA molecules, each coding for a different subunit of the hormone.

Mapping the human pulmonary surfactant-associated protein B gene (SFTP3) to chromosome 2p12-->p11.2.

Pulmonary surfactant, a lipoprotein complex is essential for normal lung function. The non-serum surfactant-associated proteins, SP-A, SP-B, and SP-C, play important roles in the biology of pulmonary surfactant. We have mapped the human SP-B gene (SFTP3) to chromosome 2, band 2p12-->p11.2 by fluorescent in situ hybridization.